The invention relates to nucleic acids which encode the heavy and light chain variable regions of a monoclonal antibody against human interleukin-5 and complementarity determining regions therefrom, and to humanized antibodies and binding proteins based upon the monoclonal antibody.
Interleukin-5 (IL-5) is a lymphokine secreted by activated T cells which is biologically active on B cells and eosinophils. Because IL-5 replaces T lymphocytes in in vitro antibody responses to thymus-dependent antigens, it was formerly called T cell replacing factor [TRF; Dutton et al., Prog. Immunol. 1:355 (1971); Schimpl et al., Nature 237:15 (1972)]. Because it also stimulates differentiation of B lymphocytes into IgM and IgG plaque-forming cells and the growth of B cell lymphomas in vitro, it has also been called B cell growth factor II [BCGFII; Takatsu et al., J Immunol. 124:2414 (1980)].
Murine IL-5 consists of 133 amino acid residues, including a signal sequence of 20 residues and three potential N-glycosylation sites. Deglycosylation does not affect the biological activity of murine IL-5 in a B cell proliferation assay [Tavernier et al., DNA 8:491 (1989)]. Human IL-5 consists of 134 amino acid residues, including a signal sequence of 19 residues and two potential N-glycosylation sites. The structures of both proteins have been described by Yokota e t al. [Proc. Natl. Acad. Sci. USA 84:7388 (1987)] and Kinashi et al. [Nature 324:70 (1986)]. The degrees of homology of murine and human IL-5 at the nucleotide and amino acid sequence level are 77 and 70%, respectively.
Both murine and human IL-5 exist as homodimers linked by disulfide bonds. Therefore, glycosylated recombinant human IL-5 migrates in SDS polyacrylamide gel electrophoresis with an apparent molecular weight of 40,000 daltons under non-reducing conditions, and 20-22,000 daltons under reducing conditions [Tsujimoto et al., J. Biochem. 106:23 (1989)].
The cloning and expression of murine IL-5 has been described, e.g., by Kinashi et al. [Nature 324:70 (1986)] and Takatsu et al. [J. Immunol. 134:382 (1985)]. Human IL-5 complementary DNA (cDNA) has been isolated using murine IL-s cDNA as a probe by Azuma et al. [Nucleic Acids Res. 14:9149 (1986)].
IL-5 has been shown to act as a maintenance and differentiation factor for eosinophils. In humans, the activity of IL-5 appears to be specific, affecting eosinophils primarily. Human IL-5 induces eosinophil precursor cells to become mature cells. Moreover, the survival of eosinophils isolated from circulating blood can be prolonged when human IL-5 is present in the culture media. Human IL-5 also stimulates cultured eosinophils to degranulate, and to release toxic proteins such as major basic protein (MBP) and eosinophil-derived neurotoxin (EDN) [Kita et al., J. Immunol. 149:629 (1992)].
It has been suggested that eosinophils kill parasites following infection and also play a significant role in inflammatory and allergic diseases [see, e.g., Sanderson, Blood 79:3101 (1992)]. Increased levels of eosinophils among circulating leukocytes have been observed following parasitic infections and in certain chronic inflammatory tissues, such as in asthmatic alveoli. Eosinophil infiltration and toxic granule release from eosinophils may play a role in tissue destruction and may aggravate the symptoms of asthma.
For example, Gleich et al. [Adv. Immunol. 39:177 (1986)] and Frigas et al. [J. Allergy Clin. Immunol. 77:527 (1986)] have shown that high-density eosinophils and eosinophil major basic protein (MBP) are associated with bronchial asthma and related tissue damage.
Recently, Coffman et al. (International Patent Application Publication No. WO/04979) have shown that antibodies against IL-5 can prevent or reduce eosinophilia which is associated with certain allergic diseases such as asthma. Monoclonal antibodies which specifically bind to and neutralize the biological activity of human IL-5 can be used for this purpose.
A monoclonal antibody against IL-5 has been reported to have a prominent effect in reversing parasite-induced eosinophilia in experimental animals [Schumacher et al, J. Immunol. 141:1576 (1988); Coffman et al., Science 245:308 (1989)], suggesting that neutralizing antibodies may be clinically useful in relieving eosinophilia-related symptoms by antagonizing IL-5. In fact, it has been reported that when rodents or monkeys bearing experimentally induced eosinophilia were treated with TRFK 5, a rat anti-mouse IL-5 monoclonal antibody, eosinophil counts in both circulation and bronchial lavage were found to return to normal levels. Thus, neutralizing monoclonal antibodies may be effective antagonists.
Because most monoclonal antibodies are of rodent origin, however, there is an increased likelihood that they would be immunogenic if used therapeutically in a human being, particularly over a long period of time. To reduce this possibility, there is a need for recombinant or xe2x80x9chumanizedxe2x80x9d antibodies against human IL-5. Such antibodies could be used for the treatment of conditions associated with eosinophilia, or for the treatment of any other condition attributable to the biological activity of IL-5.
Initial efforts to reduce the immunogenicity of rodent antibodies involved the production of chimeric antibodies, in which mouse variable regions were fused with human constant regions [Liu et al., Proc. Natl. Acad. Sci. USA 84:3439 (1987)]. It has been shown, however, that mice injected with hybrids of human variable regions and mouse constant regions develop a strong anti-antibody response directed against the human variable region. This suggests that in the human system, retention of the entire rodent Fv region in such chimeric antibodies may still give rise to human anti-mouse antibodies.
It is generally believed that CDR loops of variable domains comprise the binding site of antibody molecules, the grafting of rodent CDR loops onto human frameworks (i.e., humanization) was attempted to further minimize rodent sequences [Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)]. Studies by Kabat et al. [J. Immunol. 147:1709 (1991)] have shown that framework residues of antibody variable domains are involved in CDR loop support. It has also been found that changes in framework support residues in humanized antibodies may be required to preserve antigen binding affinity. The use of CDR grafting and framework residue preservation in a number of humanized antibody constructs has been reported, e.g., by Queen et al. [Proc. Natl. Acad. Sci. USA 86:10029 (1989)], Gorman et al. [Proc. Natl. Acad. Sci. USA 88:4181 (1991)] and Hodgson [Bio/Technology 9:421 (1991)]. Exact sequence information has been reported for only a few humanized constructs.
Although a high degree of sequence identity between human and animal antibodies has been known to be important in selecting human antibody sequences for humanization, most prior studies have used a different human sequence for animal light and heavy variable sequences. Sequences of known antibodies have been used or, more typically, those of antibodies having known X-ray structures, antibodies NEW and KOL. See, e.g., Jones et al., supra; Verhoeyen et al., supra; and Gorman et al., supra.
Methods for engineering antibodies have been described, e.g., by Boss et al. (U.S. Pat. No. 4,816,397), Cabilly et al. (U.S. Pat. No. 4,816,567), Law et al. (European Patent Application Publication No. 438 310) and Winter (European Patent Application Publication No. 239 400).
Reliance on the relatively few antibodies for which X-ray structures have been determined has led to the frequent use of different human light and heavy chain sequences from different antibodies, because although only two human Fab crystal structures are known, several human light chain crystal structures have been determined. Such an approach may require changing framework residues in the human heavy and light chains to ensure correct chain association and, therefore, limits the applicability of humanization.
There thus is a need for improved methods for making humanized antibodies that are not based upon the relatively few known crystallographic structures.
The present invention fulfills the foregoing needs by providing novel methods for the design of humanized antibodies, and specific antibody antagonists of human IL-5 and pharmaceutical compositions containing the same.
More particularly, this invention provides a method for selecting human antibody sequences to be used as human frameworks for humanization of an animal antibody comprising:
(a) comparing the heavy and light chain variable region sequences of an animal monoclonal antibody that is to be humanized with optimally-aligned sequences of the heavy and light chain variable regions of human antibodies for which sequence information is available, thereby determining the percent identities for each of the compared sequences;
(b) determining the number of ambiguities in each of such human antibody sequences;
(c) comparing Pin-region spacing of the animal antibody sequences with (i) that of each of such human antibody sequences and with (ii) those of other antibodies which have known 3-dimensional structures; and
(d) selecting the human antibody sequence which has the best combination of:
(i) low number of sequence ambiguities, and
(ii) high percent identities and similar Pin-region spacing, based on comparison to the animal antibody sequences.
This invention further provides a method for determining which variable region residues of an animal monoclonal antibody should be selected for humanization comprising:
(a) determining potential minimal and maximal residues of the animal monoclonal antibody, wherein:
(i) such minimal residues comprise CDR structural loops plus residues required to support and/or orient the CDR structural loops, and
(ii) such maximal residues comprise Kabat CDRs plus CDR structural loops plus residues required to support and/or orient the CDR structural loops plus residues which fall within about 10 xc3x85 of a CDR structural loop and possess a water solvent accessible surface of about 5 xc3x852 or greater;
(b) performing computer modeling of:
(i) a sequence of an animal monoclonal antibody which is to be humanized,
(ii) a human antibody framework sequence, and
(iii) all possible recombinant antibodies comprising the human antibody framework sequence into which the minimal and maximal residues of step (a) have been inserted,
which computer modeling is performed using software suitable for protein modeling and structural information from a structurally-characterized antibody that has a sequence most nearly identical to that of the selected human antibody framework sequence;
(c) comparing results obtained in the computer modeling of step (b); and
(d) selecting the minimal or maximal residues which produce a recombinant antibody having a computer-modeled structure closest to that of the animal monoclonal antibody.
Preferably, the human antibody framework sequence is selected as described above.
The present invention still further provides a monoclonal antibody produced by a hybridoma having the identifying characteristics of a cell line deposited under American Type Culture Collection Accession No. ATCC HB 10959, and the hybridoma itself.
This invention still further provides polypeptides comprising a heavy or light chain variable region of a monoclonal antibody which have amino acid sequences defined by SEQ ID NO: 1 and SEQ ID NO: 2, complementarity determining regions (CDRs) from such variable regions, and isolated DNAs encoding such variable regions and CDRs. These DNAs can be used to construct binding compositions, single-chain binding proteins, polypeptides which contain one or more of the CDRs and retain antigen binding activity, and recombinant antibodies comprising such CDRs, all of which are a part of this invention.
This invention still further provides pharmaceutical compositions comprising such monoclonal antibody or recombinant antibodies, binding compositions, single-chain binding proteins and polypeptides; and a physiologically acceptable carrier.